Tobacco smoking is addictive and thought to be driven by nicotine dependence (ND). For decades, data have suggested genetic bases for an increased risk of ND. More recently, several studies validated the association of rs16969968 with nicotine dependence. rs16969968 is a non-synonymous SNP, changing the 398th amino acid from aspartic acid (D) to asparagine (N) (D398N) in the CHRNA5 gene. We hypothesize that this non-synonymous variant in the CHRNA5 gene may alter the functional properties of nAChRs containing 5 subunit, ultimately altering risks for tobacco smoking and ND. To test this hypothesis, the project has four aims: 1. To generate stably transfected cell lines expressing human a4b2*- or a3b4*-nAChRs containing wild- type or variant a5 subunits and use these cell lines as model system to study the properties of major subtypes (a4b2* and a3b4*) in vitro. 2. To characterize functional and pharmacological effects of the presence of wild-type or D398N a5 subunits on a4b2*- and a3b4*-nAChR using patch-clamp technique. 3. To test whether rs16969968 is involved in smoking initiation and to test for gene-environment interactions -that is, to determine if the impact of environmental exposures on smoking behaviors are modified by the rs16969968 genotypes. 4. To establish roles of a5-containing nAChR subtypes in ND using a5 knock-out mice. The goal is to define how the presence or absence of a5 subunits in native nAChR affects acute responses to nicotine (anxiety and locomotor activity), nicotine reward (conditioned place preference, CPP), and subjective effects (nicotine discrimination). PUBLIC HEALTH RELEVANCE: Tobacco smoking and nicotine dependence is a major public health issue. Many epidemiology studies have provided compelling evidence that genetic factors play a significant role in the development of nicotine dependence. More recently, several large scale genetic studies have found that several variants in the nicotinic receptor alpha 5, alpha 3 and beta 4 genes are significantly associated with heavy smoking and nicotine dependence, including one functional polymorphism rs16969968 or D398N. This current study is designed to create model systems to examine the effects of D398N at the levels of cultured cells, animal model and human behaviors. The approaches we take are 1) To develop a cellular model system to study molecular, pharmacological and electrophysiological properties of N398D;2) To examine how genetic variants at rs16969968 interact with environmental factors and eventually lead to smoking addiction and dependence;and 3) To study the behavioral effects of these genes in animal models.